Laccase SilA from Streptomyces ipomoeae CECT 3341, a key enzyme for the degradation of lignin from agricultural residues?
ABSTRACT: The role of laccase SilA produced by Streptomyces ipomoeae CECT 3341 in lignocellulose degradation was investigated. A comparison of the properties and activities of a laccase-negative mutant strain (SilA-) with that of the wild-type was studied in terms of their ability to degrade lignin from grass lignocellulose. The yields of solubilized lignin (acid precipitable polymeric lignin, APPL) obtained from wheat straw by both strains in Solid State Fermentation (SSF) conditions demonstrated the importance of SilA laccase in lignin degradation with the wild-type showing 5-fold more APPL produced compared with the mutant strain (SilA-). Analytical pyrolysis and FT-IR (Fourier Transform Infrared Spectroscopy) confirmed that the APPL obtained from the substrate fermented by wild-type strain was dominated by lignin derived methoxyphenols whereas those from SilA- and control APPLs were composed mainly of polysaccharides. This is the first report highlighting the role of this laccase in lignin degradation.
Project description:The draft genome of Anoxybacillus sp. strain UARK-01, a novel lignin-utilizing thermophilic soil bacterium, represents the first sequence of an Anoxybacillus isolate from the United States. The genome was sequenced using the Illumina MiSeq platform, de novo assembled using SeqMan NGen, and annotated at NCBI. The genome sequence revealed genes for laccase and lignocellulose degradation enzymes.
Project description:The repertoire of redox-active enzymes produced by the marine fungus Peniophora sp. CBMAI 1063, a laccase hyper-producer strain, was characterized by omics analyses. The genome revealed 309 Carbohydrate-Active Enzymes (CAZymes) genes, including 48 predicted genes related to the modification and degradation of lignin, whith 303 being transcribed under cultivation in optimized saline conditions for laccase production. The secretome confirmed that the fungus can produce a versatile ligninolytic enzyme cocktail. It secretes 56 CAZymes, including 11 oxidative enzymes classified as members of auxiliary activity families (AAs), comprising two laccases, Pnh_Lac1 and Pnh_Lac2, the first is the major secretory protein of the fungi. The Pnh_Lac1-mediator system was able to promote the depolymerization of lignin fragments and polymeric lignin removal from pretreated sugarcane bagasse, confirming viability of this fungus enzymatic system for lignocellulose-based bioproducts applications.
Project description:Fungal laccases play important roles in the degradation of lignocellulose. Although some PoLacs have been reported in several studies, still no comprehensive bioinformatics study of the LAC family in Pleurotus ostreatus has been reported. In this study, we identified 12 laccase genes in the whole genome sequence of P. ostreatus and their physical characteristics, gene distribution, phylogenic relationships, gene structure, conserved motifs, and cis-elements were also analyzed. The expression patterns of 12 PoLac genes at different developmental stages and under different culture substrates were also analyzed. The results revealed that PoLac2 and PoLac12 may be involved in the degradation of lignin and the formation of the fruiting body, respectively. Subsequently, we overexpressed PoLac2 in P. ostreatus by the Agrobacterium tumefaciens-mediated transformation (ATMT) method. The transformants' laccase activity increased in varying degrees, and the gene expression level of PoLac2 in transformants was 2-8 times higher than that of the wild-type strain. Furthermore, the lignin degradation rate by transgenic fungus over 30 days was 2.36-6.3% higher than that of wild-type. Our data show that overexpression of PoLac2 significantly enhanced the lignin degradation of cotton-straw. To our knowledge, this study is the first report to demonstrate the functions of PoLac2 in P. ostreatus.
Project description:Lignocellulose facilitates the fungal oxidization of recalcitrant organic pollutants through the extracellular ligninolytic enzymes induced by lignin in wood or other plant tissues. However, available information on this phenomenon is insufficient. Free radical chain reactions during lignin metabolism are important in xenobiotic removal. Thus, the effect of lignin on azo dye decolorization in vivo by Echinodontium taxodii was evaluated. In the presence of lignin, optimum decolorization percentages for Remazol Brilliant Violet 5R, Direct Red 5B, Direct Black 38, and Direct Black 22 were 91.75% (control, 65.96%), 76.89% (control, 43.78%), 43.44% (control, 17.02%), and 44.75% (control, 12.16%), respectively, in the submerged cultures. Laccase was the most important enzyme during biodecolorization. Aside from the stimulating of laccase activity, lignin might be degraded by E. taxodii, and then these degraded low-molecular-weight metabolites could act as redox mediators promoting decolorization of azo dyes. The relationship between laccase and lignin degradation was investigated through decolorization tests in vitro with purified enzyme and dozens of aromatics, which can be derivatives of lignin and can function as laccase mediators or inducers. Dyes were decolorized at triple or even higher rates in certain laccase-aromatic systems at chemical concentrations as low as 10 µM.
Project description:With the growing demand for fossil fuels and the severe energy crisis, lignocellulose is widely regarded as a promising cost-effective renewable resource for ethanol production, and the use of lignocellulose residues as raw material is remarkable. Polar organisms have important value in scientific research and development for their novelty, uniqueness and diversity.In this study, a fungus Aspergillus sydowii MS-19, with the potential for lignocellulose degradation was screened out and isolated from an Antarctic region. The growth profile of Aspergillus sydowii MS-19 was measured, revealing that Aspergillus sydowii MS-19 could utilize lignin as a sole carbon source. Its ability to synthesize low-temperature lignin peroxidase (Lip) and manganese peroxidase (Mnp) enzymes was verified, and the properties of these enzymes were also investigated. High-throughput sequencing was employed to identify and characterize the transcriptome of Aspergillus sydowii MS-19. Carbohydrate-Active Enzymes (CAZyme)-annotated genes in Aspergillus sydowii MS-19 were compared with those in the brown-rot fungus representative species, Postia placenta and Penicillium decumbens. There were 701CAZymes annotated in Aspergillus sydowii MS-19, including 17 cellulases and 19 feruloyl esterases related to lignocellulose-degradation. Remarkably, one sequence annotated as laccase was obtained, which can degrade lignin. Three peroxidase sequences sharing a similar structure with typical lignin peroxidase and manganese peroxidase were also found and annotated as haem-binding peroxidase, glutathione peroxidase and catalase-peroxidase.In this study, the fungus Aspergillus sydowii MS-19 was isolated and shown to synthesize low-temperature lignin-degrading enzymes: lignin peroxidase (Lip) and manganese peroxidase (Mnp). These findings provide useful information to improve our understanding of low-temperature lignocellulosic enzyme production by polar microorganisms and to facilitate research and applications of the novel Antarctic Aspergillus sydowii strain MS-19 as a potential lignocellulosic enzyme source.
Project description:Background:As an important biomass raw material, the lignocellulose in bamboo is of significant value in energy conversion. The conversion of bamboo lignocellulose into fermentable reducing sugar, i.e. the degradation of bamboo lignocellulose, is an important step in lignocellulose conversion. However, little research has focussed on excavating the enzymes and microbes that are related to the degradation of bamboo lignocellulose, which is important for its utilisation. This study used Cyrtotrachelus buqueti (bamboo snout beetle) to evaluate the efficiency of bamboo lignocellulose degradation. Results:RNA sequencing was conducted to sequence the transcriptome of the insect before and after feeding on bamboo shoots. The expression levels of genes encoding several carbohydrate-active enzymes, such as endoglucanase (evgtrinloc27093t1 and evgtrinloc16407t0) and laccase (evgtrinloc15173t0 and evgtrinloc11252t0), were found to be upregulated after feeding. Faecal component analysis showed that the degradation efficiencies of cellulose, hemicellulose and lignin were 61.82%, 87.65% and 69.05%, respectively. After 6 days of co-culture with crude enzymes in vitro, the degradation efficiencies of cellulose, hemicellulose and lignin in bamboo shoot particles (BSPs) were 24.98%, 37.52% and 26.67%, respectively. These results indicated that lignocellulosic enzymes and related enzymes within the insect itself co-degraded bamboo lignocellulose. These finding can potentially be used for the pre-treatment and enzymatic hydrolysis of bamboo lignocellulose. Conclusion:Our results showed that intestinal digestive enzymes from C. buqueti degraded bamboo shoot lignocellulose both in vivo and in vitro. In addition, the expression levels of many carbohydrate-active enzyme (CAZyme) genes were upregulated in the transcriptome, including those for cellulase, xylanase and ligninase genes. Therefore, we proposed a scheme for applying the lignocellulolytic enzymes from C. buqueti to degrade bamboo lignocellulose using genetic, enzymatic and fermentation engineering techniques to overexpress the lignocellulolytic enzymes genes in vitro and obtain large quantities of enzymes that could efficiently degrade bamboo lignocellulose and be used for lignocellulose bioconversion.
Project description:Biological pretreatment is an important alternative strategy for biorefining lignocellulose and has attracted increasing attention in recent years. However, current designs for this pretreatment mainly focus on using various white rot fungi, overlooking the bacteria. To the best of our knowledge, for the first time, we evaluated the potential contribution of bacteria to lignocellulose pretreatment, with and without a physicochemical process, based on the bacterial strain Pandoraea sp. B-6 (hereafter B-6) that was isolated from erosive bamboo slips. Moreover, the mechanism of the improvement of reducing sugar yield by bacteria was elucidated via analyses of the physicochemical changes of corn stover (CS) before and after pretreatment.The digestibility of CS pretreated with B-6 was equivalent to that of untreated CS. The recalcitrant CS surface provided fewer mediators for contact with the extracellular enzymes of B-6. A pre-erosion strategy using a tetrahydrofuran-water co-solvent system was shown to destroy the recalcitrant CS surface. The optimal condition for pre-erosion showed a 6.5-fold increase in enzymatic digestibility compared with untreated CS. The pre-erosion of CS can expose more phenolic compounds that were chelated to oxidized Mn3+ and also provided mediators for combination with laccase, which was attributable to B-6 pretreatment. B-6 pretreatment following pre-erosion exhibited a sugar yield that was 91.2 mg/g greater than that of pre-erosion alone and 7.5-fold higher than that of untreated CS. This pre-erosion application was able to destroy the recalcitrant CS surface, thus leading to a rough and porous architecture that better facilitated the diffusion and transport of lignin derivatives. This enhanced the ability of laccase and manganese peroxidase secreted by B-6 to improve the efficiency of this biological pretreatment.Bacteria were not found useful alone as a biological pretreatment, but they significantly improved enzymatic digestion after lignocellulose breakdown via other physicochemical methods. Nonetheless, phenyl or phenoxy radicals were used by laccase and manganese peroxidase in B-6 for lignin attack or lignin depolymerization. These particular mediators released from the recalcitrance network of lignocellulose openings are important for the efficacy of this bacterial pretreatment. Our findings thus offer a novel perspective on the effective design of biological pretreatment methods for lignocellulose.
Project description:Pleurotus ostreatus is a species of white-rot fungi that effectively degrades lignin. In this study, we aimed to efficiently express the lac-2 gene of Pleurotus ostreatus in the Pichia pastoris X33 yeast strain. The enzymatic properties of recombinant yeast were determined, and its ability to degrade corn stover lignin was determined. The results showed the optimum pH values of recombinant laccase for 2,2'-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid, 2,6-dimethoxyphenol, and 2-methoxyphenol were 3.0, 3.0, and 3.5, respectively. The optimum reaction temperature was 50 °C, and it had good thermal stability and acid and alkali resistance. The degradation rate of lignin in corn stover by recombinant laccase was 18.36%, and the native Pleurotus ostreatus degradation rate was 14.05%, the difference between them is significant (p < 0.05). This experiment lays a foundation for the study of the degradation mechanism of lignin by laccase.
Project description:Multidrug resistance (MDR) that occurs in cancer cells constitutes one of the major reasons for chemotherapy failure. The main molecular mechanism of MDR is overexpression of protein transporters from the ATP-binding cassette (ABC) superfamily, such as ABCB1 (multidrug resistance protein 1 (MDR1), P-glycoprotein). At the expense of ATP hydrolysis, ABCB1 pumps a diverse range of substrates (including anticancer drugs) out of the cell, thereby reducing their intracellular concentration. In the present study, the ability of two patented disiloxanes (SILA-409 and SILA-421) to reverse drug resistance in human colon adenocarcinoma cell lines LoVo and LoVo/Dx was investigated. It was demonstrated that both compounds in concentrations of 0.5-1 µM strongly increased the sensitivity of LoVo/Dx cells to doxorubicin. By means of an accumulation test in which rhodamine 123 was used as an ABCB1 substrate analogue, both organosilicon compounds were also shown to inhibit ABCB1 transport activity. The intracellular accumulation of doxorubicin was also increased, and more drug entered the cellular nuclei of resistant cells in the presence of the studied compounds. In conclusion, both SILA-409 and SILA-421 were demonstrated to be effective MDR reversal agents in resistant human colon cancer cells.
Project description:Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH(2)). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH(2) extended the reactions catalyzed by these enzymes to the production of H(2)O(2), the oxidation of Mn(2+), the reduction of Fe(3+), and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.